title: Cooperation of p53 loss of function and v-Ha-ras in transformation of mouse keratinocyte cell lines.
abstract: We previously demonstrated that after transduction with the v-Ha-ras oncogene and grafting onto nude mouse hosts, primary epidermal keratinocytes with a null mutation in the p53 gene form tumors with increased growth rates and predisposition to malignant conversion relative to p53 wild-type keratinocytes (Weinberg WC, et al., Cancer Res 54:5584-5592, 1994). To further explore the cooperation between p53 loss of function and activation of the ras oncogene, cell lines were established from the normal epidermises of newborn and adult p53-null mice, and parallel subclones were reconstituted with the p53val135 temperature-sensitive mutant. Reconstituted lines C, G, N, and V demonstrated functional p53 transcriptional activator activity at the wild-type-permissive temperature of 32 degrees C, compared with the hygromycin-selected control line X and parental p53-null lines NHK4 and AK1b. Hygromycin-selected subclones, but not the parental lines, made normal skin in vivo; all cell lines made carcinomas after introduction of v-Ha-ras, independent of p53 status. These cell lines were compared in vitro at 32 degrees C to maximize the amount of p53val135 in the wild-type conformation. Expression of v-Ha-ras did not consistently alter p53-mediated transcriptional activity, suggesting tat ras acts downstream or independently of p53. No correlation was observed between p53-mediated transcriptional activity and in vitro growth rates, colony formation after exposure to ultraviolet light, or suppression by normal neighboring keratinocytes. However, keratinocyte cell lines devoid of p53 and expressing v-Ha-ras formed colonies in soft agar; this was blocked at 32 degrees C in all cell lines reconstituted with p53val135. These keratinocyte lines provide a model for exploring the role of p53 and the interaction of p53 and ras in keratinocyte transformation.
authors.affiliation: Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, MD 20892-4255, USA.
authors.forename: C G
authors.initials: CG
authors.lastname: Azzoli
authors.forename: M
authors.initials: M
authors.lastname: Sagar
authors.forename: A
authors.initials: A
authors.lastname: Wu
authors.forename: D
authors.initials: D
authors.lastname: Lowry
authors.forename: H
authors.initials: H
authors.lastname: Hennings
authors.forename: D L
authors.initials: DL
authors.lastname: Morgan
authors.forename: W C
authors.initials: WC
authors.lastname: Weinberg
mesh_terms: D000818:Animals; D002455:Cell Division / Q000235:genetics; D002470:Cell Survival / Q000528:radiation effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000473:pathology; D017690:Cell Transplantation; D000078404:Epidermal Cells; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D005786:Gene Expression Regulation; D016158:Genes, p53 / Q000235:genetics / Q000502:physiology*; D011905:Genes, ras / Q000502:physiology*; D015603:Keratinocytes / Q000378:metabolism / Q000473:pathology / Q000502:physiology* / Q000528:radiation effects; D051379:Mice; D008819:Mice, Nude; D014466:Ultraviolet Rays